Engineered pertactin variants for vaccine use

ABSTRACT

The present invention is related with the field of Biomedicine. It comprises the engineering of the Pertactin protein (Prn) and using it as part of bacterial vaccines, and more precisely, as part of acellular vaccines against  Bordetella pertusis . The engineered Prn molecules comprise on their structure polimorfisms from different  B. pertussis  strains, and induce immune responses with protective capacity and opsonophagocytic activity when assayed as vaccines, higher than that generated by other pre-existing vaccines. The engineered Prn variants of the present invention are applicable in human and veterinary medicine.

TECHNICAL FIELD

The present invention is related with the field of Biomedicine. Itcomprises the engineering of the Pertactin protein (Prn) and using it aspart of bacterial vaccines, and more precisely, as part of acellularvaccines against Bordetella pertusis. The engineered Prn moleculescomprise on their structure polimorfisms from different B. pertussisstrains, and induce immune responses with increased protective capacityand opsonophagocytic activity when assayed as vaccines, higher than thatgenerated by other pre-existing vaccines.

BACKGROUND OF THE INVENTION

Whooping Cough or Pertussis is an acute, highly infectious respiratorydisease caused by the Bordetella pertussis bacterium, a microorrganismformerly isolated by Bordet and Gengou in 1906 [Bordet, J. and O.Gengou. Ann Inst Pasteur (Paris), 1906. 20: p. 731-41]. Recently, theannual morbidity of infections throughout the world was estimated in48.5 millions. The disease is particularly severe in children with lessthan six months of age, with 90% of the casualties being associated tothis ethereal group (300,000-400,000) [Crowcroft, N. S., et al. LancetInfect Dis, 2003. 3(7): p. 413-8].

Several vaccines are available against B. pertussis, distributed in twomain groups according to their type: cellular vaccines, and, morerecently, acellular vaccines. Vaccination has dramatically decreased theincidence of the disease, moving it from children towards teenager andadult populations. Several studies have shown teenagers as the majorreservoir for B. pertussis and the main source for spreading of thisdisease among partially protected children. Therefore, whooping coughremains as an unsolved health problem, demanding the development of newvaccines for a better control of the epidemics and re-emergentoutbreaks, and possibly to eradicate this disease in endemic regions[Cherry, J. D. Pediatrics, 2005. 115(5): p. 1422-7; Singh, M. and K.Lingappan, Chest, 2006. 130(5): p. 1547-53]. The Bordetella generaincludes nine species, four of them being associated to infections inmammals (B. holmesii, B. bronchiseptica, B. parapertussis and B.pertussis), the last two being responsible for infections in humans[Mattoo, S., et al. Front Biosci, 2001. 6: p. E168-86]. Most of theirvirulence factors are regulated at transcriptional level by atwo-components system denominated BvgA/S (Bordetella virulence genesActivator/Sensor) [Stibitz, S., et al. Nature, 1989. 338(6212): p.266-9]. Among them, the most relevant factors are the Pertussis toxins(PT), the tracheal colonization factor, adenylate cyclase; and adhesinsfilamentous Phytohemagglutinin (PHA), Fimbriae (Fim) and Pertactin(Prn), the latter being the focus of the present invention.

Prn is an outer membrane protein belonging to the family of type Vautotransporter proteins. It is characterized by catalyzing its owntransportation through the bacterial outer membrane [Henderson, I. R.Trends Microbiol, 2000. 8(12): p. 534-5]. The mature Prn is a protein of68 kDa in B. bronchiseptica [Henderson, I. R. Infect Immun, 2001. 69(3):p. 1231-43.], 69 kDa in B. pertussis [Charles, I. G., et al. Proc NatlAcad Sci USA, 1989. 86(10): p. 3554-8] and 70 kDa in B. parapertussis[Li, L. J., et al. Mol Microbiol, 1991. 5(2): p. 409-17], respectively.Its structure consists on 16 paralel strands forming a β helix and atransversal section in V form [Emsley, P., et al. Nature, 1996.381(6577): p. 90-2.]. Numerous loops protrude from this helicoidal core.One of them is the Arg-Gly-Asp triplete (RGD), a motif associated totissue adherence [Leininger, E., et al. Infect Immun, 1992. 60(6): p.2380-5; Emsley, P., et al. Nature, 1996. 381(6577): p. 90-2]. Thepresence of this motif and numerous proline-rich regions are related toPrn functions during adhesion. Experiments have shown that the Prn canmediate adhesion to cells of the respiratory epithelium [Everest, P., etal. Microbiology, 1996. 142 (Pt 11): p. 3261-8]. Nevertheless, assays onthe inhibition by human sera of B. pertussis adhesion to A549 culturedcells (alveolar human epithelium) did not evidence Prn as a crucialelement during that process under the tested conditions [Rodriguez, M.E., et al. FEMS Immunol Med Microbiol, 2006. 46(1): p. 39-47].

The Prn protein is part of acellular vaccines composed of three or morecomponents. Acellular vaccines can be composed of: 1) one component ofPT, 2) two components: PT and PHA, 3) three components: PT, PHA and Prn,and 4) five components, including the three components previouslymentioned and also the Fimbriae 2 (Fim2) and Fimbriae 3 (Fim3) proteins.In humans, the levels of the anti-Prn, anti-Fim2 and anti-PT antibodiescorrelate with protection levels against the disease [Cherry, J. D., etal. Vaccine, 1998. 16(20): p. 1901-6; Storsaeter, J., et al. Vaccine,2003. 21(25-26): p. 3542-9].

The active immunization with Prn of B. pertussis and B. bronchisepticainduces a specific antibody response against Prn, conferring protectionin different animal models [Charles, I. G., et al. Eur J Immunol, 1991.21(5): p. 1147-53; Roberts, M., et al. Vaccine, 1992. 10(1): p. 43-8].Similarly, the passive administration of anti-Prn monoclonal antibodies(MAbs) protected mice in the model of respiratory challenge [King, A.J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95]. Protectionlevels in mice subjected to the intranasal challenge assay (INCA) wereincreased by adding Prn to vaccines containing PT and PHA [Guiso, N., etal. Vaccine, 1999. 17(19): p. 2366-76]. It has been recently shown thatPrn is the only component of acellular vaccines which generates anantibody response of such a level that correlates to theopsonophagocytic activity [Hellwig, S. M., et al. J Infect Dis, 2003.188(5): p. 738-42]. In spite of efficacious vaccines and the wellestablished vaccination programs available, whooping cough is stillendemic in regions of America, Europe and Asia, being considered as are-emergent disease [Raguckas, S. E., et al. Pharmacotherapy, 2007.27(1): p. 41-52]. One of the hypotheses trying to explain thisphenomenon is based on the loss of efficacy, due to appearance ofresistant strains [Mooi, F. R et al. Emerg Infect Dis, 2001. 7(3 Suppl):p. 526-8]. Prn is one of the most polymorphic proteins in B. pertussis.It contains two variable regions designated as region 1 (R1) and 2 (R2),respectively, with repetitive amino acid sequences rich in prolineGly-Gly-X-X-Pro (GGXXP) and Pro-Gln-Pro (PQP) motifs. The R1 region islocated in the protruding loop proximal to the aminoterminal sequence(N-terminal) and near to the RGD motif, while the R2 region is locatednear to the carboxyl terminal end (C-terminal) [Hijnen, M., et al.Infect Immun, 2004. 72(7): p. 3716-23]. Up to 12 different variants ofPrn (Prn1, Prn2, Prn3 . . . Prn12) have been identified in B. pertussis,as shown in the database of the National Center for BiotechnologyInformation of the United States of America (NCBI). Strains bearing thePrn1, Prn2 and Prn3 are distributed worldwide. Numerous straincharacterization studies, either retrospective or of strains currentlycirculating, were carried out in American, European, Asian andAustralian regions and showed a tendency towards a progressivepersistence of Prn2 strains over Prn1 strains, the Prn2 strainspredominating in most of the countries studied [Mooi, F. R., et al.Infect Immun, 1998. 66(2): p. 670-5; Cassiday, P et al. J Infect Dis,2000. 182(5): p. 1402-8; Weber, C. et al. J Clin Microbiol, 2001.39(12): p. 4396-403; Hallander, H. O., et al. J Clin Microbiol, 2005.43(6): p. 2856-65; van Amersfoorth, S. C., et al. J Clin Microbiol,2005. 43(6): p. 2837-43; Byrne, S, et al. BMC Infect Dis, 2006. 6: p.53].

Current differences in the amino acid sequence of Prn between cellular(DTPc) or acellular (DPTa) vaccines and circulating strains is one ofthe factors supporting the hypothesis of the efficacy loss of vaccinesavailable, due to the appearance of new strains. Studies in populationsvaccinated with DPTc or DTPa, and non-vaccinated populations, inNetherlands and Italy indicated that these types of vaccines protectbetter against circulating strains similar to the vaccine strain [Mooi,F. R., et al. Infect Immun, 1998. 66(2): p. 670-5; Mastrantonio, P., etal. Microbiology, 1999. 145 (Pt 8): p. 2069-75]. In agreement with thesefindings, it was shown in the mice model that vaccination with DPTcdifferentially protects against strains bearing Prn1 and Prn2,indicating that changes in the Prn R1 region can confer resistancelevels [King, A. J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95].However, massive studies stratifying B. pertussis strains according tocountry of origin, vaccination status, and type of vaccines (DPTc andDPTa), did not show significant differences in the frequencies of prn,ptxC, ptxA or tcfA2 alleles for circulating strains and vaccinationprograms [van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6):p. 2837-43]. The high prevalence of Prn2 strains in many countries isindicative of the favored transmission of these strains by means stillunraveled, although the findings mentioned above hardly link the originof new variants to vaccination. Remarkably, in the above mentioned study[van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p.2837-43], the three clinically isolated strains bearing allelles similarto that of the vaccines used were found only in non-vaccinated children.Either casual or not, it suggests that Prn1 strains are favored inniches devoid of specific immunity. On the other hand, the recentidentification of a phage infecting Bordetella (BPP-1) by using Prn asprimary receptor, suggested that variations in this protein might betriggered by selective pressures other than those imposed by the immunesystem [Liu, M., et al. Science, 2002. 295(5562): p. 2091-4]. Thepossible influences of both phenomena, together with other unknownfactors leading to harmonized variations in B. pertussis, are notexcluded.

The evolution of Pertussis epidemiology has been simulated by amathematical model, integrating the incidence of the disease and thepathogen's transmission independently [Aguas, R., et al. Lancet InfectDis, 2006. 6(2): p. 112-7]. This model predicts that regular boostingdoses would not be capable of eliminating the severity grades of thedisease, observed in current epidemics. It is highly probable that thisshould be caused by the short lifespan of the protection conferred bythe available acellular vaccines (4-12 years), and also the variabilityof the immune response and the different types of vaccines. This modelpredicts as the most optimistic scenario that where vaccines couldgenerate an immunity superior to the natural one, a paradigm stillunreached by the cellular and acellular vaccines available.

The main purpose of the present invention resides on the contribution todevelop more efficacious acellular vaccines against Whooping Cough. Themain work preceding the present invention were based on administeringimmunogenic preparations obtained by mixing Prn proteins (Nicole Guisoet al., WO 01/90143 A2 y US 2006/0008474 A1) or synthetic peptides ofthe Prn R1 region (Frederik Mooi et al., WO 02/00695 A2). Therefore, thedevelopment of more efficacious acellular vaccines is an importantproblem to prevent Whooping Cough.

DETAILED DESCRIPTION OF THE INVENTION

This invention contributes to solve the above mentioned problems, andcomprises the engineering of the prnA gene, coding for the outermembrane protein of B. pertussis denominated Pertactin (Prn). Thisinvention suffices the needs evidenced in the state of the art, makingpossible obtaining different variants of engineered Prn, in such a waythat they comprise in their structure two different polymorphic domainsof the Prn R1 region. The versatility of the invention also covers theengineering of new Prn molecules, additionally comprising three or moredifferent polymorphic domains of the Prn R1 region.

Is subject of the present invention a polynucleotidic sequence codingfor an engineered Prn protein, wherein said protein comprises up to thefirst 300 amino acids proximal to the N-terminal end of a natural,mature Prn of a given type (PrnX300), and an aminoacidic sequencecomprising up to 620 amino acids proximal to the C-terminal end of anatural, mature Prn of given type (PrnY620), resulting in an engineeredPrnX300-PrnY620Prn protein.

In the context of the present invention, the term ‘engineered Prn’refers to a protein resulting from coupling, adjacently or not, of afragment comprising up to the first 300 amino acids proximal to theN-terminal end of a given natural, mature Prn protein, to anotherfragment comprising the last 620 amino acids proximal to the C-terminalend of a natural, mature Prn protein.

The new Prn engineered variants are obtained by molecular mutagenesis,by adjacent coupling of sequences comprising up to the first 300 aminoacids proximal to the N-terminal end of a natural, mature Prn of a giventype, to sequences comprising up to the last 620 amino acids proximal tothe C-terminal end of a natural, mature Prn of a given type. The newvariants of engineered Prn comprise sequences from the same or differenttype of Prn in a single molecule, without affecting the protectiveimmune response.

In a preferred embodiment of the present invention, different variantsof Prn engineered variants are obtained, encoded by the nucleic acidsequences identified as SEQ ID Nr. 1-SEQ ID Nr. 6. Highly significantlyprotection levels and opsonophagocytic activities were obtained byimmunizing mice with the different variants of engineered Prn, higherthan those obtained with natural Prn molecules formulated alone orcombined in mixes. The immune response generated with the engineered Prnwas equally effective against strains expressing different types of Prn.

In a preferred embodiment of the present invention, the fragmentcomprising the first 300 amino acids proximal to the N-terminal end of anatural, mature Prn of a given type, named PrnX300, corresponds to Prnfrom the genera Bordetella. In another preferred embodiment of thepresent invention, this fragment corresponds to Prn molecules from B.pertussis or B. parapertussis, preferably Prn1, Prn2 and Prn3 variantsof B. pertussis.

In a preferred embodiment of the invention, the las 620 amino acidsproximal to the C-terminal end of a natural, mature Prn of a given type,named PrnY620, corresponds to Prn from the genera Bordetella. In anotherpreferred embodiment of the present invention, this fragment correspondsto Prn molecules from B. pertussis or B. parapertussis, preferably Prn1,Prn2 and Prn3 variants of B. pertussis.

The polynucleotidic sequence of the present invention codes for apolypeptidic sequence comprising any possible combination of Prn typesin the format PrnX300-PrnY620.

The amino acid sequences PrnX300 and PrnY620 coded by the polynucleotidesequence of the present invention are adjacently coupled, or by usingthe amino acid sequences IDNATWVMTDN or IDNATWVMTDNIDNATWVMTDN.

In the present invention, the amino acid sequences PrnX300 and PrnY620can be devoid of repetitive sequences, preferably of GGXXP and PQPsequences of the R1 and R2 regions. The evidences supporting this designare the following: the Region 1 (R1), comprising the repetitive sequenceGGXXP is weakly recognized by human and rabbit sera, indicating that itis not an immunodominant region [Hijnen, M., F. R. Mooi, et al. (2004).Infect Immun 72(7): 3716-23]. On the other hand, recent work reportedPrn mutants where the repetitive GGXXP and PQP sequences or regionscontaining these sequences were deleted. GGXXP deletions did not affectthe physicochemical properties of the mutant Prn molecules obtained, asevidenced in the similar methods used for expression and purification ofmutant and non-mutant Prn proteins [Hijnen, M., P. G. van Gageldonk, etal. (2005). Protein Expr Purif 41(1): 106-12]. Similarly, deletions ofthe GGXXP sequences did not significantly affected structuralproperties, since Prn molecules mutated in R1 were well recognized byMAbs generated against conformational epitopes in natural Prn molecules,and also not recognized by anti-GGXXP MAbs directed against linear GGXXPepitopes. Additionally, it was observed that certain mutations inside R1can enhance the binding capacity to certain MAbs against conformationalepitopes. Finally, there were evidences indicating that R1 (GGXXP) andR2 (PQP) form a single epitope [Hijnen, M., R. de Voer, et al. (2007).Vaccine 25(31): 5902-14]

In another preferred embodiment of the present invention, the saidpolynucleotidic sequence codes for an engineered Prn, wherein said aminoacid PrnX300 and PrnY620 sequences comprise heterologous peptides ableto function as T helper cell epitopes isolated from Diphtheria, Tetanus,the hepatitis B virus (HBV), Polioviruses, Vaccinia, the humanimmunodeficiency virus (HIV) or the human Influenza virus. It is wellknown among people skilled in the art that the immune response against agiven antigen can be enhanced by including this type of epitopes.

An additional preferred embodiment of the present invention comprisesthe polynucleotidic sequences according to claim 1, wherein saidpolynucleotide sequences could be optimized for optimal codon usage, toincrease the expression of the encoded protein in bacteria, yeast,insect or mammalian cells. The resulting increase in the expression ofthe molecules encoded by recombinant procedures is widely know by peopleskilled in the art in this particular technical field.

In another preferred embodiment, the new protein subject of the presentinvention can be one of the multiple components of a new combinedvaccine, emphasizing that none of the precedent inventions comprisedobtaining the minimal number of molecular entities satisfying theexisting requirements of this technical field.

Finally, the demands for vaccine preparations able to generatecross-protection between B. pertussis and B. parapertussis are more thanevident in the state of the art. The present invention also comprisesgenerating engineered Prn molecules comprising in a single structuredifferent polymorphic regions of different Bordetella species, based onthe high homology levels existing between Prn proteins of the differentBordetella species.

Unexpectedly, the engineered Prn subject of the present invention wasnot only capable of inducing an effective immune response againstdifferent Prn1- and Prn2-expressing B. pertussis strains, but alsogenerated antibody responses more effective than that generated by othernon-engineered recombinant Prn proteins, as evidenced in the micerespiratory challenge model and the opsonophagocytic assay.Surprisingly, the immune response induced by the engineered Prn wassuperior to that induced by an equimolar mix of Prn1 and Prn2(Prn1+Prn2).

Vaccine compositions made by mixing different Prn proteins of the sameor different species, although covering polymorphisms, lead to technicaldifficulties associated to the new production processes, such as theundesired increase in the concentration of contaminants and theproductive inconsistency between lots. This is an essential aspect todevelop combined vaccines, composed of multiple antigens with quitedifferent characteristics, which can compromise the systemicimmunogenicity of the formulation. On the other hand, it is expectedthat strategies based on synthetic peptides of the R1 region could leadto vaccines less effective than vaccines currently available, byexcluding other epitopes present in the natural Prn from the antigen,relevant to develop a protective response.

To meet this unsolved requirement of this technical field, the presentinvention comprises a pharmaceutical composition comprising one or moreengineered Prn, coded by polynucleotidic sequences from Claims 1 to 13,in amounts sufficient to generate humoral and cellular immune responseseffective against Bordetella species, when administered throughimmunization procedures in mammals, and preferably, in humans. In apreferred embodiment of the present invention, the pharmaceuticalcomposition comprising one or more Prn engineered variants generateshumoral and cellular immune responses effective against B. pertussis.

It is also the aim of the present invention a life or attenuated vaccinecomprising one or more Prn engineered variants, coded by sequences fromClaims 1 to 13, wherein said Prn engineered variants are expressed inthe outer membrane of the life or attenuated organism. In this live orattenuated vaccine, said polynucleotidic sequences from Claims 1 to 13are included in a plasmid vector or a bacterial chromosome.

In another embodiment of the present invention, said polynucleotidicsequences from Claims 1 to 13, which code for Prn engineered variants,are included in a vector for expression in mammalian cells. In anotherembodiment of the present invention, said expression vector whichcontains the polynucleotidic sequences from Claims 1 to 13 is the basisfor a nucleic acids vaccine.

In another embodiment of the invention, the polypeptidic sequences codedby said polynucleotidic sequences from Claims 1 to 13, can be used todetect Bordetella infections. Is also the aim of the present invention adiagnostic kit to detect the presence or absence of antibodies againstBordetella, comprising polypeptidic sequences coded by thepolynucleotidic sequences referred on Claims 1 to 13.

BRIEF DESCRIPTION OF THE FIGURES

is FIG. 1. Protection experiment in Balb/c mice vaccinated withdifferent recombinant Prn engineered variants. Strains of B. pertussisTohama I (Prn1) and the clinical isolate CH53 (Prn2) were used aschallenge. Barrs represent the mean logarithm of the reduction of viablebacterial cells in lungs.

FIG. 2. Opsonophagocytosis mediated by sera from Balb/c mice vaccinatedwith the different recombinant Prn engineered variants. The chart showsthe difference of fluorescence (phycoerithrin, PE) in arbitrary units(AU) of cells stained with fluorescein isothiocyanate (FITC) in twoincubation conditions (PE 4° C.-PE 37° C.).

FIG. 3. Humoral IgG immune response against Prn1 and Prn2CCPrn1generated in mice immunized with plasmids expressing the Prn1, Prn2,Prn2CCPrn1 and Prn2CLPrn1 engineered variants.

DETAILED DESCRIPTION OF THE EMBODIMENTS/EXAMPLES Example 1 Constructionof Vectors for the Intracellular Expression in Escherichia Coli of theDifferent Prn Engineered Variants and its Purification

The prnA1 and prnA2 genes from Bordetella strains B. pertussis Tohoma I(Prn1) and CH53 (Prn2) were amplified by Polymerase Chain Reaction (PCR)from genomic DNA by using the previously reported oligonucleotides 1 and2 [Hijnen, M., P. G. van Gageldonk, et al. (2005). Protein Expr Purif41(1): 106-12].

The fragments obtained were cloned into the vector pET-28^(a) (Novagen)using the sites Nde I and BamH I. The Prn engineered variants wereobtained by using the reverse PCR method previously reported by Imai andco-workers in 1991 [Imai, Y., et al. Nucleic Acids Res, 1991. 19(10): p.2785]. Nucleotides used to amplify the different polynucleotidicsequences are shown in Table 1 The oligonucleotide pair 1,2 was used tolinearize vector pET28aprn1 and pETaprn2, corresponding to Prn1 andPrn2, respectively. The DomR1 fragments were obtained by amplificationwith oligonucleotides 3 and 4. Additionally, this region was amplifiedby using the nucleotide pairs 3,5 and 3,6 to add sequences coding forthe short and long linkers, respectively. The conditions used for PCRamplification of the fragments used in the present invention aresummarized in Table 2.

TABLE 1Oligonucleotides used for amplification of the different sequences.Oligonucleotide Result of the PCR Number name Sequence 5′→3′amplification 1 pET28aprn1 1401- AGCGTGGAGCTCGCCCALinearized vector with 30 LinVect GTCGATCGTCGAG blunt ends 2pET28aprn1 1431- GGAGCCCGATACGTCCA 60 LinVect CGCCATACCAGCC 3pET28aprn1 1975- GTCAAGGCCGGCAAGCT Domain R1 (DomR1) of 97 DomR1 GGTCGCany type of Prn 4 pET28aprn1 1431- GGAGCCCGATACGTCCA 53 DomR1 CGCCAT 5pET28aprn1 1431- ATCGACAACGCCACCTG Amplifies DomR1 from 53 DomR1 CC-NtGGTCATGACGGACAACG any type of Prn, also TCAAGGCCGGCAAGCTGadding a linker of 11 GTCGC amino acids to the N- term. 6pET28aprn1 1431- ATCGACAACGCCACCTG Amplifies DomR1 from 53 Dom R1 CL-NtGGTCATGACGGACAACA any type of Prn, also TCGACAACGCCACCTGGadding a linker of 22 GTCATGACGGACAACGT amino acids to the N-CAAGGCCGGCAAGCTG term.

TABLE 2 Conditions for PCR amplification of the different fragments usedin the present invention Size of the Oligonucl Hybridation TemplateExtension Polymerase No. of Amplific. amplific. pair temp (° C.). DNA(μg) time (min) (Units) cycles product product (bp) 1, 2 65 pET28aprn17.5 Pfx (2.5) 5 Vector 7370 (1) Lineal 1, 2 65 pET28aprn2 7.5 Pfx (2.5)5 Vector 7385 (1) Lineal 3, 4* 67 pET28aprn1 0.6 Pfu (2.5) 30 DomR1 567(0.1) prn1 3, 4* 67 pET28aprn2 0.6 Pfu (2.5) 30 DomR1 582 (0.1) prn2 3,5* 67 DomR1 prn1 0.6 Pfu (2.5) 30 CC- 600 (0.1) DomR1 prn1 3, 6* 67DomR1 prn1 0.6 Pfu (2.5) 30 CL- 633 (0.1) DomR1 prn1 3, 5* 67 DomR1 prn20.6 Pfu (2.5) 30 CC- 620 (0.1) DomR1 prn2 3, 6* 67 DomR1 prn2 0.6 Pfu(2.5) 30 CL- 648 (0.1) DomR1 prn2 *Phosphorylated oligonucleotides, CC:Short linker, CL: Long linker

The linearized pET28aprn1 and pET28aprn2 vectors, obtained by reversePCR, were ligated to the different fragments coding for domainscontaining region 1 from Prn1 and Prn2. In these vectors, the newengineered genes are under the transcriptional control of the T7inducible promoter. Clones bearing the correct sequences were introducedinto the BL21-Codonplus(DE3)-RP E. coli strain, for the expression ofthe corresponding proteins as inclusion bodies [Hijnen, M., et al.Protein Expr Purif, 2005. 41(1): p. 106-12].

The expression levels of the recombinant Prn1 and Prn2, as well as forthe other variants, reached between 15 and 20% of total proteins, asevidenced by densitometry in polyacrylamide gels stained with Coomassieblue.

The different proteins were purified by suspending the bacterial pastefor each variant in rupture buffer (at a cell concentration of 100mg/mL) and cells were lysed with ultrasound. The cellular pellets weresolubilized in 8 M Urea and fractionated by Sodium Dodecyl Sulphatepolyacrylamide gel electrophoresis (SDS-PAGE, 12.5%). The gel wasstained by reverse Zinc-Imidazol staining, and the slice containing theband corresponding to the protein of interest was passed through astainless steel mesh of 100 μm in the presence of extraction buffer. Theprotein was further extracted, and renatured and concentrated byultrafiltration through an Amicon concentration cell, with a membrane of50 kDa, and the final concentration was determined by the Bicinchoninicacid method. No contaminants were detected by assaying 15 μg of eachprotein purified from the analytical SDS-PAGE gels stained withCoomassie blue, evidencing that protein preparations were more than 95%pure. The characteristics of the different constructs and the Prnengineered variants obtained are summarized in Table 3.

TABLE 3 Characteristics of the different Prn constructs and Prnengineered variants Linker between Name of the Characteristics ofPlasmid name DomR1 engineered Prn Prn type the engineered Prn pET28aprn1No Prn1 1 — pET28aprn2 No Prn2 2 — pETprn DomR1 No Prn1-Prn2 1, 2 Nt . .. DomR1(Prn1)- (1-2) DomR1(Prn2) . . . Ct pETprn DomR1 IDNATWVMTPrn1-CC-Prn2 1, 2 Nt . . . DomR1(Prn1)-CC- (1-CC-2) DN DomR1(Prn2) . . .Ct pETprn DomR1 IDNATWVMT Prn1-CL-Prn2 1, 2 Nt . . . DomR1(Prn1)-CL-(1-CL-2) DNIDNATWV DomR1(Prn2) . . . Ct MTDN pETprn DomR1 — Prn2-Prn1 1,2 Nt . . . DomR1(Prn2)- (2-1) DomR1(Prn1) . . . Ct pETprn DomR1IDNATWVMT Prn2-CC-Prn1 1, 2 Nt . . . DomR1(Prn2)-CC- (2-CC-1) DNDomR1(Prn1) . . . Ct pETprn DomR1 IDNATWVMT Prn2-CL-Prn1 1, 2 Nt . . .DomR1(Prn2)-CL- (2-CL-1) DNIDNATWV DomR1(Prn1) . . . Ct MTDN

Example 2 Active Immunization, Antibody Response and Protection in aMice Model

Mice were immunized with 0.2 μg or 0.02 μg of the recombinant Prn1 andPrn2, PBS, an equimolar mix of Prn1 and Prn2 (Prn1+Prn2), and six of thePrn engineered variants (shown in Table 3). All the proteins wereadministered formulated in alum. Doses corresponded to 1/40 and 1/400fractions of the dose commonly employed in humans (Infanrix®, 8 μg).Mice were immunized by the subcutaneous route, with a volume of 100 μL.Sera from the immunized mice were evaluated by an ELISA typeimmunoenzymatic assay. The antibody titers reached mean values from1.2×10³ to 4.6×10⁴. The mean values of the titers for corresponding tothe highest doses significantly differed from the titers reached withthe lowest doses used, for all the cases (p<0.05, Kruskal Wallis-Dunns).No differences were observed in the antibody response generated withPrn1, Prn2 or the equimolar mix Prn1+Prn2. Similarly, there were nodifferences between the mean titers of the different Prn engineeredvariants. Surprisingly, the titers obtained with the Prn engineeredvariants were significantly higher than those generated by thenon-engineered recombinant Prn proteins (p<0.01, Kruskal Wallis-Dunns).

The strain Tohama I (Prn1) and the clinical isolate CH53 (Prn2) wereused for the intranasal challenge. Bacteria were cultures in platescontaining Bordet-Gengou-Agar media (Sigma) supplemented with 1%glycerol and 14% defibrinated goat blood. Plates were incubated for 24 hat 37° C. and the resulting colonies were suspended in Stainer-Scholtemedium at a 10⁸ cells/mL concentration. This suspension was used for theintranasal challenge. Mice immunized were challenged 15 days after thelast immunization, by instillation of 50 μL of the bacterial suspension(5×10⁶ cells). Five days after challenge, mice were sacrificed and lungsaseptically extracted and homogenized to measure the bacterial burden[Denoel, P., et al. Vaccine, 2005. 23(46-47): p. 5333-41]. The differentvariants showed protection levels significantly higher than thenon-vaccinated controls (p<0.001). Unexpectedly, the engineered Prnvariants showed higher protection levels when compared with therecombinant Prn proteins or the equimolar Prn1+Prn2 mix for both strains(p<0.001).

The Prn engineered variants showed similar protection levels againstboth strains at the lowest administered dose, an effect unattained withthe Prn1 or Prn2 proteins. These results evidence that these Prnengineered variants bear immunological properties different from, andsuperior to, those showed by the recombinant Prn1 and Prn2 proteinsassayed both separately or as equimolar mixes (FIG. 1).

Example 3 Opsonophagocytic Activity in Sera

The opsonophagocytic activity mediated by anti-Prn sera has been shownas a crucial parameter in the response of people vaccinated withacellular vaccines [Hellwig, S. M., et al. J Infect Dis, 2003. 188(5):p. 738-42]. The present invention shows that the different Prnengineered variants were capable of inducing antibodies resembling theseproperties. The opsonophagocytic activity was studied by the previouslymentioned method, adapted to the mice model. Strains Tohama I and CH53of B. pertussis were grown in Bordet-Gengou-Agar and the cells stainedwith FITC (2×10⁶ colony-forming units). Afterwards, the labeled bacteriawere opsonized for 30 min at 37° C., in a plate shaker, with sera frommice immunized with the recombinant Prn1, Prn2, Prn1+Prn2 and twovariants of the engineered Prn proteins (Prn2-CC-Prn1 and Prn2-CL-Prn1).During the adhesion step, the opsonized bacteria and the non-opsonizedcontrol were incubated with polymorphonuclear cells (PMN). Samples werefurther divided into two equal subgroups of cells, one incubated foranother 45 min at 4° C. and the other at 37° C. All the samples werefinally incubated for another 30 min at 4° C. with the goatanti-mouse-PE labeled conjugate. Samples were analyzed by flow cytometry(PARTEC PAS III). The fluorescence intensities of green- and red-stainedcells incubated at 4° C. were used as adhesion controls. The differenceof red fluorescence in green-stained cells was used to evidence thephagocytic activity mediated by sera.

The Prn engineered variants showed opsonophagocytic activity (FIG. 2).Surprisingly, there were significant differences only in among thegroups immunized with the Prn engineered variants when compared withmice inoculated with PBS (p<0.05, Kruskal Wallis-Dunns). Theopsonophagocytic activity of sera generated by the recombinant,non-engineered Prn protein alone or in combination reached values 6-foldhigher than the PBS control, although these differences were notsignificant. Finally, these results evidenced that the Prn engineeredvariants are able to induce antibodies with significant opsonophagocyticactivity, irrespective of the type of Prn present in the bacterium.

Example 4 Construction of Vectors for the Expression in Mammals of PrnEngineered Variants and Evaluation of the Humoral Immune ResponseGenerated

The genes prnA1 and prnA2, and the gene variants prn2CCprn1 andprn2CLprn1, were amplified by PCR from their respective expressionvectors (see Table 3) by using the previously reported oligonucleotides1 and 2 [Hijnen, M., P. G. van Gageldonk, et al. (2005). Protein ExprPurif 41(1): 106-12]. In this case, the oligonucleotide 1 was modified,substituting the Nde I by a BamH I restriction site. The fragmentsobtained were cloned into the BamH I restriction site of the pAEC-SPE3plasmid vector [Herrera A M, Rodriguez E G, et al. (2000) BBRC, 279,548-551]. This vector is designed for the extracellular expression ofantigens in mammalian cells. The resulting constructs were purified byusing the commercial plasmid DNA purification kit Endo-free plasmid Gigakit (Qiagen). Groups of female 6-to-7-week-old Balb/c mice wereimmunized thrice with 100 μg of DNA in 100 μL of PBS, at three-weekintervals by the intraperitoneal route. The control group was immunizedwith the empty vector without insert (pAEC-SPE3). Fifteen days after thelast immunization, mice were sacrificed and blood collected for theevaluation of sera. The specific IgG antibody responses were evaluatedby the ELISA technique, at a 1/1000 dilution and coating plates withequimolar amounts of the Prn1 (2 μg/mL) and Prn2CCPrn1 (2.4 μg/mL)proteins. As shown in FIG. 3, animals immunized with the differentplasmids expressing Prn1, Prn2, Prn2CCPrn1 and Prn2CLPrn1 generatedspecific IgG antibody responses, significantly higher (p<0.001) thananimals immunized with the empty pAEC-SPE3 vector. Similarly to seragenerated in mice immunized with protein and alum (data not shown), serafrom immunized mice preferentially recognized the Prn engineered variantPrn2CCPrn1 (p<0.05), more than the natural Prn1 protein, what could bedue to a better exposure of the shared epitopes in Prn2CCPrn1 than inPrn1.

1. An isolated polynucleotide sequence coding for a pertactin (Prn)engineered protein, wherein said isolated polynucleotide sequence codesfor up to the first 300 amino acids proximal to the N-terminal end of agiven type of natural, mature Prn (PrnX300) and an amino acid sequencecomprising up to the last 620 amino acids proximal to the C-terminal endof a given type of natural, mature Prn (PrnY620), resulting in anengineered PrnX300-PrnY620 pertactin, and wherein the polynucleotidesequence comprises SEQ ID NO:
 4. 2. The isolated polynucleotide sequenceaccording to claim 1, wherein said PrnX300 amino acid sequence comprisesPrn sequences from the Bordetella genera.
 3. The isolated polynucleotidesequence according to claim 2, wherein said PrnX300 amino acid sequencecomprises Prn sequences from B. pertussis or B. parapertussis.
 4. Theisolated polynucleotide sequence according to claim 3, wherein saidPrnX300 amino acid sequence comprises Prn sequences from Prn1, Prn2 andPrn3 of B. pertussis.
 5. The isolated polynucleotide sequence accordingto claim 1, wherein said PrnY620 amino acid sequence comprises Prnsequences from the Bordetella genera.
 6. The isolated polynucleotidesequence according to claim 5, wherein said PrnY620 amino acid sequencecomprises Prn sequences from B. pertussis or B. parapertussis.
 7. Theisolated polynucleotide sequence according to claim 3, wherein saidPrnY620 amino acid sequence comprises Prn sequences from Prn1, Prn2 andPrn3 of B. pertussis.
 8. The isolated polynucleotide sequence accordingto claim 1, wherein said polynucleotide sequence codes for a polypeptidecomprising any possible combination of Prn of any type in the formatPrnX300-PrnY620.
 9. The isolated polynucleotide sequence according toclaim 1, wherein said PrnX300 and PrnY620 amino acid sequences arecoupled adjacently, or by means of IDNATWVMTDN or IDNATWVMTDNIDNATWVMTDNamino acid sequences.
 10. The isolated polynucleotide sequence accordingto claim 1, wherein said PrnX300 and PrnY620 amino acid sequences aredevoid of repetitive sequences, and more precisely, devoid of GGXXP andPQP repetitive sequences from Prn regions R1 and R2.
 11. The isolatedpolynucleotide sequence according to claim 1, wherein said PrnX300 andPrnY620 amino acid sequences additionally comprise peptides with Thelper epitopes.
 12. (canceled)
 13. The isolated polynucleotide sequenceaccording to claim 1, that has an optimized codon usage for optimalexpression in bacteria, yeast, insect or mammalian cells.
 14. Apharmaceutical composition comprising a polynucleotide sequenceaccording to claim
 1. 15. A pharmaceutical composition according toclaim 14, wherein said pharmaceutical composition generates effectivehumoral and cellular immune responses against B. pertussis. 16.(canceled)
 17. (canceled)
 18. An isolated expression vector comprisingan isolated polynucleotide sequence according to claim 1, wherein saidsequence codes for engineered Prn molecules.
 19. (canceled)
 20. A methodto detect B. pertussis or B. parapertussis infections, comprising theuse of a polypeptide sequence according to claim
 1. 21. A diagnostic kitfor detection of B. pertussis or B. parapertussis, wherein saiddiagnostic kit comprises a polynucleotide sequence according to claim 1.22. The isolated polynucleotide sequence according to claim 11, whereinsaid peptides are obtained from Diphtheria, Tetanus, HBV, Poliovirus,Vaccinia, HIV or Influenza virus.